Sophisticated and varied genetic tools exist for manipulating the genomes of established model microorganisms, such as Escherichia coli and Saccharomyces cerevisiae. However, for many other microorganisms of biotechnological interest, only exceedingly basic genetic tools are available, which makes it difficult to evaluate and optimize such microorganisms for medical, chemical, or industrial applications.
For example, genetic tools are lacking for the genus Clostridium, which includes Gram-positive, spore-forming, anaerobic bacteria. Species such as Clostridium difficile, Clostridium botulinum, and Clostridium perfringens are pathogenic and/or have important medical applications. Additionally, species such as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium celluloyticum, Clostridium ljungdahlii, Clostridium butyricum, and Clostridium autoethanogenum ferment sugars, biomass, and gases to produce various biofuels and biochemical products.
Existing genetic tools for Clostridium, such as ClosTron (Heap, J Microbiol Meth, 70:452-464, 2007), allele coupled exchange (ACE) (Heap, Nucleic Acids Res, 40: e59, 2012), and counter selection markers (Ng, PLoS ONE, 8: e56051, 2013; Al-Hinai, Appl Environ Microbiol, 78: 8112-8121, 2012; Cartman, Appl Environ Microbiol, 78: 4683-4690, 2012; WO 2010/084349), allow only rudimentary genetic manipulation compared to genetic tools for model microorganisms like Escherichia coli and Saccharomyces cerevisiae. Moreover, the genetic tools that do exist often require multiple steps to achieve the desired modification, cumbersome mutant screening processes, and fickle transformation steps. Accordingly, there is a strong need for robust genetic tools and methods for manipulating the genomes of non-model microorganisms, such as Clostridium bacteria.